In recent years, a nitrile hydratase has been discovered which is an enzyme having the nitrile-hydrating activity to convert a nitrile group of various compounds to an amide group by hydration, and a number of microorganism strains producing the above-mentioned enzyme have been disclosed. In order to produce an amide compound from a nitrile compound using a nitrile hydratase on an industrial scale, it is important to reduce the production costs for this enzyme in the total production costs for producing the amide compound. More specifically, it is necessary to increase the activity value in a unit weight of the preparation obtained from the enzyme production.
As a method for increasing the activity value by increasing the amount of the enzyme in the enzyme preparation, attempts have already been made to clone the gene encoding the above-mentioned enzyme for the purpose of expressing a large amount of the enzyme through genetic engineering methods.
For example, there are produced a plasmid expressing a large number of the Pseudonocardia thermophila-derived nitrile hydratase in the transformant and a transformant strain transformed with the plasmid. In addition, it has been made possible to produce a nitrile hydratase by means of these transformant strains, and to produce a corresponding amide compound by bringing the transformant strain or the nitrile hydratase obtained therefrom into contact with the nitrile compound (see Patent Document 1).
On the other hand, when high activation of enzyme molecule itself can be achieved, the activity value of the enzyme preparation can be further enhanced.
Attempts have heretofore been made to search for a nitrile hydratase variant with improved substrate specificity, enzyme stability or the like by introducing mutation into a specific amino acid residue in the amino acid sequence of the nitrile hydratase without damaging its activity (see Patent Document 2 to 4).
Furthermore, a nitrile hydratase variant derived from Rhodococcus rhodochrous has been disclosed in Patent Documents 5 and 6.